Common Laboratory Techniques in Experimental Pharmacology.pdf

1. Common Laboratory
Techniques in
Experimental Pharmacology
Dr. Santosh B. Dighe, M. Pharm, Ph.D., LL.B. , PGDAW
Associate Professor and Head , Department of Pharmacology
Pravara Rural College of Pharmacy, Loni

2. Content of Presentation
Animal handling
Routes of Drug Administration
Blood collection techniques
Anesthesia
Euthanasia

3. Handling and restraint
: Good handling and restraint is the most important technique for correct
administration .There are two styles of manual restraint
1. Double handed manual restraint 2. Single handed restraint

6. Rats are generally fed a diet
containing low fiber, protein and
fat. Rat rooms are usually
maintained at 30-70% relative
humidity and a temperature at 18-
26⁰C .
Rats should be acclimatized to handling to reduce stress.
Blood can be collected from several sites in the rat
including tail vein, retro-orbital sinus, vena-cava or cardiac
puncture. Can receive oral, IP, IM, IV

7. Mice
The mouse and human genomes are about 85% the
same, and those similarities have made the mouse a
powerful model for studying human biology and
disease.
Handling, blood collection, and drug
administration are same as rats.

8. Various routes of drug administration

9. Enteral Route of administration
Placement of drug directly into any part of the GIT
It could be Oral, Sublingual ,Intragastric gavage, or Rectal.

10. 1-Oral
Swallowing a drug through mouth, It may be done by
adding desired drug to the drinking water or to the food
The oral route is economical, convenient, relatively safe,
and some animals can be trained to cooperate voluntarily,
depending on the compound being administered
This route is not preferable since it inaccurate.

11. is the administration of fluids directly
into the lower esophageal or stomach.
Gavage is often used in research
settings, instead of mixing substances in
water or food, to ensure accurate
dosing of animals.
A small, curved, metal tube, usually
with a ball on the end (feeding needle)
is often used with small rodents.
Entrance may normally be obtained
without anesthesia using ordinary hand
restraint and the ball prevents trauma
to the esophagus and oral cavity.
2- Intra-gastric gavage

12. Procedure for gastric gavage in rodents:
1. Fill the syringe with the appropriate volume of material and attach the needle.
2. Restrain the animal by the scruff. Place the tip or ball of the needle into the animal’s
mouth. Slide the tip gently past the back of the tongue.
3.The needle should slide easily down the esophagus, if properly placed. DO NOT FORCE!!! If
any resistance is met, remove the needle and reinsert. Do not aspirate. Once the needle is
properly placed, administer the material.
➢To make sure that the tube is in the esophagus and not in the trachea, dip the end of the
tube into a beaker containing water (bubbling indicates wrong position).
➢ A safe volume to gavage rats and mice is 10 ml gavage solution per kg body weight.

13. Parenteral routes of administration
o Routes other than Enteral are called Parenteral routes of administration
o Parenteral administration methods typically produce the highest bioavailability of
substances because these methods avoid the first-pass effect of hepatic metabolism.
1 Intravenous (IV) directly in the vascular system through a vein
2 Intraperitoneal (IP) - injected into the abdominal cavity
3 Intramuscular (IM) injected into a muscle
4 Subcutaneous (SC) injected under the skin
5 Intradermal (ID) - injected between the layers of the skin

14. 6 intracerebral(IC)- injected into the brain
7 Epidural - injected into the epidural space of the spinal cord
8 Intranasal- sprayed into the nose for absorption across the nasal mucous
9 Inhalation: Inspiration through nose or mouth
10 Intra-articular: injection directly into the joint space

15. 1-Subcutaneous (SC) injections
The best spot to inject Subcutaneously is the loose skin on the back of the neck
A mouse may easily be injected by one person, whereas a rat may require
restraint by one person and injection by the other
Not suitable for large volumes. Suitable for some insoluble suspensions

16. Procedure
oLift the skin over the back to form a tent.
oPlace the mouse on the wire lid so it can hang on . Scruff
the skin over the back and tent it up.
oInsert the needle at the tent base, Hold the needle
parallel to the animal’s body to also avoid puncturing
underlying structures.
oAspirate to ensure that the needle has not entered a
blood vessel.
oWithdraw the needle and then press the skin to seal the
needle’s exit hole in the skin and to prevent the fluid
from leaking out.

17. 2-Intra dermal (ID) injections
oIt is very difficult in the mouse due to thin skin
oUsing a needle of 29G or smaller it is recommended
oThe mouse is first anesthetized , the fur or hair is removed from an
area on the back , ventral abdomen, hind footpad , which is wiped
with 70% ethanol on a gauze sponge or swab.
oThe skin is held tautly with thumb and index finger and the needle
is inserted, bevel up and at a shallow angle, just under the superficial
layer of epidermis.

18. oThe volume should be <0.05 ml per site.
oResistance should be felt both as the needle is
advanced and as the compound is injected.
oA hard bleb will be seen upon successful
intradermal injection of even a small quantity
of fluid.
oIf multiple sites are injected , adequate
separation is necessary to prevent coalescing
of lesions.

19. 3-Intraperitoneal (IP) injections
oCommonly used in rats and mice since muscle mass is so
small and veins are difficult to find
oRapid absorption (almost as fast as IV) due to large
peritoneal surface
oIP administration results in a faster absorption into the
vasculature than SC administration
oA mouse may easily be injected by one person, whereas a rat
might require restraint by one person and injection by the
other
oVolume of vehicle ranging between 2 ml/kg to 10 ml/kg

20. oThe injection site is usually on the animal’s lower abdominal quadrant
o Insert the needle at approximately 45 degree angle
oThere are three points that you need to pay attention:
position/ angel / draw back.
ofirst the position of injection is in the abdomen, not too high,
not too low, if too high, liver may be hurt, if too low, bladder
may be hurt.
oSecond, the angle should be about 45 degree.
oThird, after the syringe needle has been in the abdomen,
before injection, you should draw back the stylet to see if can
draw out something if not , you can go on
oIf there is draw out of blood or urine, that shows you have
fail

21. 4- Intravenous (IV) injections
ois the most efficient means of delivering
substances to animals because it bypasses the
need for solute absorption
oTechnically difficult, and the use of a restraining
device with appropriate size for the animal to be
injected, is often required
oPerformed in mice and rats, use the lateral tail
vein located on either side of the tail

22. o The tail vein is difficult to find that’s
why mouse is often placed under a
heat lamp to promote peripheral
vasodilation
o Suitable for large volumes. Must
inject slowly

23. 5-Intramuscular (IM) injections
oIntramuscular injections result in uniform and rapid absorption of
substances, because of the rich vascular supply
oNot recommended in mice and small species due to their small
muscle mass
oSmaller volumes are administered intramuscularly than for
subcutaneous delivery
oSuitable for aqueous or specialized depot preparations

24. Procedure
o One person restrains the mouse by the scruff method with one hand
and steadies the leg to be injected with the other
o The second person , aspirates and ,
bevel up. Direct the needle caudally
(toward tail) if using the caudal thigh
muscles or cranially (toward head) if
using the quadriceps It is very
important to avoid injuring the sciatic
nerve.

25. o Aspirate to ensure that you have not entered a blood vessel.
o If no blood is seen, slowly inject the material

26. Injection site and volume in Rodents
Route Maximum needle size Optimal volume Site
Gavage Mice: 20 Gauge, (3.8cm)
length
Rat: 16 Gauge, (7.6cm) length
5 mL/kg (to 20 mL/kg ) intragastric
IV 25 Up to 5 mL/kg tail or Retro
vein
-orbital
Sc. 25 Maximum of 5 mL/kg
per site
Intrascapular
(Scruff),
neck, Flank
IM 25 -27 Maximum of 0.05
mL/kg per site
caudal thigh ,
quadriceps
muscles
IP 23 -25 Maximum of 10 mL/kg Lower ventral
quadrants
ID 29G or smaller <0.05ml per site back,ventral abdomen, foot

27. Blood Collection

48. ANAESTHESIA
It is a state of controlled temporary loss of sensation or awareness that or
awareness that is induced for medical purpose.
REVERSIBLE AND controlled loss of consciousness constant security
for the animal.
Minimal physiological and psychological trauma optimal condition for
surgery .

50. EUTHANASIA
Euthanasia is the act of including animal death in an animal. sacrificing the experimental
animal after use by gentle procedure causing minimum of physical and mental suffering is
called euthanasia
Methods of euthanasia:-
Chemical methods:
Inhalant agent: example- chloroform, carbon monoxide (co), carbon dioxide (CO2) etc.
Injectable agent: example- Barbiturates, chloral hydrate, potassium chloride , ethanol etc.
Physical methods:
•Decapitation.
•Dislocation of the neck.
•Blow of the head.
•Electrical current.